Semiconductor nanocrystal probes for biological applications and process for making and using such probes

ABSTRACT

A semiconductor nanocrystal compound and probe are described. The compound is capable of linking to one or more affinity molecules. The compound comprises (1) one or more semiconductor nanocrystals capable of, in response to exposure to a first energy, providing a second energy, and (2) one or more linking agents, having a first portion linked to the one or more semiconductor nanocrystals and a second portion capable of linking to one or more affinity molecules. One or more semiconductor nanocrystal compounds are linked to one or more affinity molecules to form a semiconductor nanocrystal probe capable of bonding with one or more detectable substances in a material being analyzed, and capable of, in response to exposure to a first energy, providing a second energy. Also described are processes for respectively: making the semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and treating materials with the probe.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/714,284 filed Feb. 26, 2010 which is a divisional of U.S. patentapplication Ser. No. 11/566,998 filed Dec. 5, 2006, which is acontinuation of U.S. patent application Ser. No. 10/155,918 filed May24, 2002 (now abandoned) which is a continuation of U.S. patentapplication Ser. No. 09/781,621 filed Feb. 12, 2001, now U.S. Pat. No.6,727,065 issued Apr. 27, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/259,982 filed Mar. 1, 1999, now U.S. Pat. No.6,207,392 issued Mar. 27, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 08/978,450 filed Nov. 25, 1997, now U.S.Pat. No. 5,990,479 issued Nov. 23, 1999, which applications areincorporated herein by reference.

The invention described herein arose in the course of, or under,Contract No. DE-AC03-SF00098 between the United States Department ofEnergy and the University of California for the operation of the ErnestOrlando Lawrence Berkeley National Laboratory. The Government may haverights to the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to semiconductor nanocrystal probes forbiological applications wherein the probes include a plurality ofsemiconductor nanocrystals capable of providing a detectable signal inresponse to exposure to energy.

2. Description of the Related Art

Fluorescent labeling of biological systems is a well known analyticaltool used in modern bio-technology as well as analytical chemistry.Applications for such fluorescent labeling include technologies such asmedical (and non-medical) fluorescence microscopy, histology, flowcytometry, fluorescence in-situ hybridization (medical assays andresearch), DNA sequencing, immuno-assays, binding assays, separation,etc.

Conventionally, such fluorescent labeling involves the use of an organicdye molecule bonded to a moiety which, in turn, selectively bonds to aparticular biological system, the presence of which is then identifiedby excitation of the dye molecule to cause it to fluoresce. There are anumber of problems with such an analytical system. In the first place,the emission of light of visible wavelengths from an excited dyemolecule usually is characterized by the presence of a broad emissionspectrum as well as a broad tail of emissions on the red side of thespectrum, i.e., the entire emission spectrum is rather broad. As aresult, there is a severe limitation on the number of different colororganic dye molecules which may be utilized simultaneously orsequentially in an analysis since it is difficult to eithersimultaneously or even non-simultaneously detect or discriminate betweenthe presence of a number of different detectable substances due to thebroad spectrum emissions and emission tails of the labeling molecules.Another problem is that most dye molecules have a relatively narrowabsorption spectrum, thus requiring either multiple excitation beamsused either in tandem or sequentially for multiple wavelength probes, orelse a broad spectrum excitation source which is sequentially used withdifferent filters for sequential excitation of a series of probesrespectively excited at different wavelengths.

Another problem frequently encountered with existing dye molecule labelsis that of photostability. Available fluorescent molecules bleach, orirreversibly cease to emit light, under repeated excitation (10⁴-10⁸cycles of absorption/emission). These problems are often surmounted byminimizing the amount of time that the sample is exposed to light, andby removing oxygen and/or other radical species from the sample.

In addition, the probe tools used for the study of systems by electronmicroscopy techniques are completely different from the probes used forstudy by fluorescence. Thus, it is not possible to label a material witha single type of probe for both electron microscopy and forfluorescence.

It would, therefore, be desirable to provide a stable probe material forbiological applications preferably having a wide absorption band andcapable of providing a detectable signal in response to exposure toenergy, without the presence of the large red emission tailscharacteristic of dye molecules (thereby permitting the simultaneous useof a number of such probe materials, each, for example, emitting lightof a different narrow wavelength band) and/or capable of scattering ordiffracting radiation. It would also be equally desirable to provide asingle, stable probe material which can be used to image the same sampleby both light and electron microscopy.

SUMMARY OF THE INVENTION

The invention comprises a semiconductor nanocrystal compound capable oflinking to one or more affinity molecules to form a semiconductornanocrystal probe. The semiconductor nanocrystal compound comprises oneor more semiconductor nanocrystals and one or more first linking agents.The one or more semiconductor nanocrystals are capable of providing adetectable signal in response to exposure to energy, wherein such aresponse may include emission and/or absorption and/or scattering ordiffraction of the energy to which the one or more semiconductornanocrystals are exposed. In addition to or as an alternative toproviding a detectable signal, the one or more semiconductornanocrystals may transfer energy to one or more proximal structures inresponse to exposure to energy. The one or more first linking agentshave a first portion linked to one or more semiconductor nanocrystalsand a second portion capable of linking either to one or more secondlinking agents or to one or more affinity molecules.

The invention further comprises a semiconductor nanocrystal probe formedeither by (1) linking one or more of the above described semiconductornanocrystal compounds to one or more affinity molecules; or (2) linkingone or more of the above described semiconductor nanocrystal compoundsto one or more second linking agents and linking the one or more secondlinking agents to one or more affinity molecules, wherein the one ormore affinity molecules are capable of bonding to one or more detectablesubstances in a material. As a result, the semiconductor nanocrystalprobe, in one embodiment, is capable of absorbing energy from either aparticle beam or an electromagnetic radiation source (of broad or narrowbandwidth), and is capable of emitting detectable electromagneticradiation in a narrow wavelength band when so excited; while in anotherembodiment the amount of energy from either a particle beam or anelectromagnetic radiation source (of broad or narrow bandwidth) which isabsorbed, or scattered, or diffracted by the semiconductor nanocrystalprobe, is detectable, i.e., the change in absorption, scattering, ordiffraction is detectable. In yet another embodiment, the semiconductornanocrystal probe is capable of receiving energy transferred from aproximal source and/or transferring energy to one or more proximalstructures in response to exposure to energy.

The invention also comprises a process for making the semiconductornanocrystal compound and for making the semiconductor nanocrystal probecomprising the semiconductor nanocrystal compound linked to one or moreaffinity molecules capable of bonding to one or more detectablesubstances. The semiconductor nanocrystal probe of the invention isstable with respect to repeated excitation by light, or exposure toelevated temperatures, or exposure to oxygen or other radicals.

The invention further comprises a process for treating a material, suchas a biological material, to determine the presence of a detectablesubstance in the material, which comprises a step of contacting thematerial to be treated, with the semiconductor nanocrystal probe, anoptional step of removing from the material the semiconductornanocrystal probes not bonded to the detectable substance, and then astep of exposing the material to energy from, for example, either anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam. The presence of the detectable substance in the materialis then determined by a step of detecting the signal provided by thesemiconductor nanocrystal probe in response to exposure to energy. Thismay be accomplished, for example, either by measuring the absorption ofenergy by the semiconductor nanocrystal probe and/or detecting theemission of radiation of a narrow wavelength band by the semiconductornanocrystal probe and/or detecting the scattering or diffraction ofenergy by the semiconductor nanocrystal probe, indicative (in eithercase) of the presence of the semiconductor nanocrystal probe bonded tothe detectable substance in the material.

The invention further comprises a process for treating a material, suchas a biological material with a semiconductor nanocrystal probe which isused to transfer energy to one or more proximal structures. This processcomprises a step of contacting the material to be treated, with thesemiconductor nanocrystal probe, an optional step of removing from thematerial portions of the semiconductor nanocrystal probe not bonded tothe detectable substance, and then a step of exposing the material toenergy from, for example, either an electromagnetic radiation source (ofbroad or narrow bandwidth) or a particle beam. This is followed by astep of energy transfer from the semiconductor nanocrystal probe to oneor more proximal structures which may, in response to the energytransfer, either provide a detectable signal, undergo chemical orconformational changes, or transfer energy to one or more secondproximal structures.

The use of the semiconductor nanocrystal probe in the treatment of amaterial to either provide a detectable signal or transfer energy to aproximal structure may be applied to a plurality of medical andnon-medical biological applications. Exemplary applications of thesemiconductor nanocrystal probe include: use as a detector of substanceson the surface or interior of cells in flow cytometry; use in aplurality of methods for detecting nucleic acid sequences byhybridization, such as fluorescence in-situ hybridization (particularlywhen the semiconductor nanocrystal probe has been modified in apolymerase chain reaction); or use to transfer energy which may causethe release of a cytotoxic molecule or transfer of heat energy, eitherof which may result in death of specifically targeted cells. Another useof the semiconductor nanocrystal probe is as a precursor which istreated to synthetic steps which result in a modified semiconductornanocrystal probe (as in the case of modification by polymerase chainreaction).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of the semiconductor nanocrystal compound ofthe invention.

FIG. 2 is a block diagram of the semiconductor nanocrystal probe of theinvention.

FIG. 3 is a block diagram showing the affinity between a detectablesubstance and the semiconductor nanocrystal probe of the invention.

FIG. 4 is a flow sheet illustrating the process of forming thesemiconductor nanocrystal probe of the invention.

FIG. 5 is a flow sheet illustrating a typical use of the semiconductornanocrystal probe of the invention in detecting the presence of adetectable substance in a material such as a biological material.

FIG. 6 is a flow sheet illustrating a typical use of the semiconductornanocrystal probe of the invention in transferring energy to a proximalstructure.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises a semiconductor nanocrystal compound capable oflinking to either one or more second linking agents or to one or moreaffinity molecules, and capable of providing a detectable signal inresponse to exposure to energy. The semiconductor nanocrystal compound,in turn, comprises: (1) one or more semiconductor nanocrystals, eachcapable of providing a detectable signal in response to exposure toenergy; and (2) one or more first linking agents, each having a firstportion linked to the semiconductor nanocrystal and a second portioncapable of linking either to one or more second linking agents or to oneor more affinity molecules.

The invention also comprises the above described semiconductornanocrystal compound linked to one or more affinity molecules (througheither one or more first linking agents, or through one or more secondlinking agents which are in turn linked to one or more first linkingagents) to form a semiconductor nanocrystal probe capable of bonding toone or more detectable substances and capable of providing a detectablesignal in response to exposure to energy. Treatment of a material(typically a biological material) with the semiconductor nanocrystalprobe, and subsequent exposure of this treated material to energy, asdescribed above, to determine the presence of the detectable substancewithin the material, will result in the semiconductor nanocrystal in thesemiconductor nanocrystal probe bonded to the detectable substanceproviding a detectable signal. This detectable signal, such as a changein absorption and/or emission of electromagnetic radiation of a narrowwavelength band and/or scattering or diffraction may signify (in eitherinstance) the presence in the material, of the detectable substancebonded to the semiconductor nanocrystal probe.

The invention also comprises a process for making the semiconductornanocrystal compound, and a process for making the semiconductornanocrystal probe comprising the semiconductor nanocrystal compoundlinked to one or more affinity molecules capable of bonding to one ormore detectable substances.

The invention further comprises a process for treating a material, suchas a biological material, to determine the presence of one or moredetectable substances in the material which comprises: (1) contactingthe material with the semiconductor nanocrystal probe, (2) (optionally)removing from the material the semiconductor nanocrystal probes notbonded to the detectable substance, (3) exposing the material to energy(such as the above-described electromagnetic energy source or particlebeam), to which, the semiconductor nanocrystal is capable of providing aresponse, signifying the presence of the semiconductor nanocrystal probebonded to the detectable substance in the material, and (4) detectingthe signal provided by the semiconductor nanocrystal in thesemiconductor nanocrystal probe.

The invention further comprises a process for treating a material, suchas a biological material, using a semiconductor nanocrystal probe, whichcomprises: (1) contacting the material with the semiconductornanocrystal probe, (2) (optionally) removing from the material thesemiconductor nanocrystal probes not bonded to the detectable substance,(3) exposing the material to energy (such as an electromagnetic energysource or particle beam) capable of causing a transfer of energy fromone or more semiconductor nanocrystal probes to one or more proximalstructures in response to exposure to energy, and (4) transferringenergy from one or more semiconductor nanocrystal probes to one or moreproximal structures.

a. Definitions

By use of the terms “nanometer crystal” or “nanocrystal” herein is meantan organic or inorganic crystal particle, preferably a single crystalparticle, having an average cross-section no larger than about 20nanometers (nm) or 20×10⁻⁹ meters (200 Angstroms), preferably no largerthan about 10 nm (100 Angstroms) and a minimum average cross-section ofabout 1 nm, although in some instances a smaller average cross-sectionnanocrystal, i.e., down to about 0.5 nm (5 Angstroms), may beacceptable. Typically the nanocrystal will have an average cross-sectionranging in size from about 1 nm (10 Angstroms) to about 10 nm (100angstroms).

By use of the term “semiconductor nanocrystal” is meant a nanometercrystal or nanocrystal of Group II-VI and/or Group III-V semiconductorcompounds capable of emitting electromagnetic radiation upon excitation,although the use of Group IV semiconductors such as germanium orsilicon, or the use of organic semiconductors, may be feasible undercertain conditions.

The term “radiation,” as used herein, is meant to includeelectromagnetic radiation, including x-ray, gamma, ultra-violet,visible, infra-red, and microwave radiation; and particle radiation,including electron beam, beta, and alpha particle radiation.

The term “energy” is intended to include electromagnetic radiation,particle radiation, and fluorescence resonance energy transfer (FRET).As used herein, the term “first energy” is meant the energy to which asemiconductor nanocrystal is exposed. Use of the term “second energy” ismeant energy provided by a semiconductor nanocrystal, within asemiconductor nanocrystal compound or within a semiconductor nanocrystalprobe, in response to exposure to a first energy. It should be notedthat different nanocrystals, when exposed to the same “first energy”,may respectively provide “second energies” which differ from oneanother, and the use of the term “second energy”, when used inconnection with a plurality of semiconductor nanocrystals will beunderstood to refer to either second energies which are the same or to aplurality of different second energies.

By the use of the term “energy transfer” is meant the transfer of energyfrom one atom or molecule to another atom or molecule by eitherradiative or non-radiative pathways.

The term “proximal source” is meant an atom, a molecule, or any othersubstance which is capable of transferring energy to and/or receivingenergy transferred from another atom or molecule or any other substance.

The term “proximal structure” as used herein may be an atom, a molecule,or any other substance (e.g. a polymer, a gel, a lipid bilayer, and anysubstance bonded directly to a semiconductor nanocrystal probe) which iscapable of receiving energy transferred from another atom or molecule orother substance (including a semiconductor nanocrystal probe).

By use of the term “a narrow wavelength band”, with regard to theelectromagnetic radiation emission of the semiconductor nanocrystal, ismeant a wavelength band of emissions not exceeding about 40 nm, andpreferably not exceeding about 20 nm in width and symmetric about thecenter, in contrast to the emission bandwidth of about 100 nm for atypical dye molecule, with a red tail which may extend the band widthout as much as another 100 nm. It should be noted that the bandwidthsreferred to are determined from measurement of the width of theemissions at half peak height (FWHM), and are appropriate in the rangeof 200 nm to 2000 nm.

By use of the term “a broad wavelength band”, with regard to theelectromagnetic radiation absorption of the semiconductor nanocrystal ismeant absorption of radiation having a wavelength equal to, or shorterthan, the wavelength of the onset radiation (the onset radiation isunderstood to be the longest wavelength (lowest energy) radiationcapable of being absorbed by the semiconductor nanocrystal), whichoccurs near to, but at slightly higher energy than the “narrowwavelength band” of the emission. This is in contrast to the “narrowabsorption band” of dye molecules which occurs near the emission peak onthe high energy side, but drops off rapidly away from that wavelengthand is often negligible at wavelengths further than 100 nm from theemission.

The term “detectable signal: as used herein, is meant to includeemission by the semiconductor nanocrystal of electromagnetic radiation,including visible or infrared or ultraviolet light and thermal emission;and any other signal or change in signal emanating from thesemiconductor nanocrystal evidencing scattering (including diffraction)and/or absorption in response to exposure of the semiconductornanocrystal to radiation.

By use of the term “detectable substance” is meant an entity or group orclass of groups, the presence or absence of which, in a material such asa biological material, is to be ascertained by use of the semiconductornanocrystal probe of the invention.

By use of the term “affinity molecule” is meant the portion of thesemiconductor nanocrystal probe of the invention which comprises anatom, molecule, or other moiety capable of selectively bonding to one ormore detectable substances (if present) in the material (e.g.,biological material) being analyzed.

The use of the term “small molecule” as used herein (for either anaffinity molecule or a detectable substance) is any atom or molecule,inorganic or organic, including biomolecules, having a molecular weightbelow about 10,000 daltons (grams/mole).

By use of the term “linking agent” is meant a substance capable oflinking with one or more semiconductor nanocrystals and also capable oflinking to one or more affinity molecules or one or more second linkingagents.

By use of the term “first linking agent” is meant a substance capable ofeither (1) linking with one or more semiconductor nanocrystals, and alsocapable of linking to one or more affinity molecules; or (2) linkingwith one or more semiconductor nanocrystals and also capable of linkingto one or more second linking agents.

By use of the term “second linking agent” is meant a substance capableof linking to one or more affinity molecules and also capable of linkingto one or more linking agents.

Use of the term “three-dimensional structure” herein is meant to defineany structure, independent of shape, which is greater than 10 nm inthickness along the three mutually perpendicular principle axes of thestructure.

Use of the term “substructure” herein is meant one of two or moreportions of a three-dimensional structure.

The terms “link” and “linking” are meant to describe the adherencebetween the one or more affinity molecules and the one or moresemiconductor nanocrystals, either directly or through one or moremoieties identified herein as linking agents (including second linkingagents between the linking agent and the affinity molecule). Theadherence may comprise any sort of bond, including, but not limited to,covalent, ionic, hydrogen bonding, van der Waals forces, or mechanicalbonding, etc.

The terms “bond” and “bonding” are meant to describe the adherencebetween the affinity molecule and the detectable substance. Theadherence may comprise any sort of bond, including, but not limited to,covalent, ionic, or hydrogen bonding, van der Waals forces, ormechanical bonding, etc.

The term “semiconductor nanocrystal compound”, as used herein, isintended to define one or more semiconductor nanocrystals linked to oneor more first linking agents and capable of linking to either one ormore second linking agents or to one or more affinity molecules, whilethe term “semiconductor nanocrystal probe” is intended to define asemiconductor nanocrystal compound linked to one or more affinitymolecules.

The term “glass” as used herein is intended to include one or moreoxides of silicon, boron, and/or phosphorus, or a mixture thereof, aswell as the further optional inclusion of one or more metal silicates,metal borates or metal phosphates therein.

b. The Semiconductor Nanocrystals

The semiconductor nanocrystals useful in the practice of the inventioninclude nanocrystals of Group II-VI semiconductors such as MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixed compositionsthereof; as well as nanocrystals of Group III-V semiconductors such asGaAs, InGaAs, InP, and InAs and mixed compositions thereof. As mentionedabove, the use of Group IV semiconductors such as germanium or silicon,or the use of organic semiconductors, may also be feasible under certainconditions. The semiconductor nanocrystals may also include alloyscomprising two or more semiconductors selected from the group consistingof the above Group III-V compounds, Group II-VI compounds, Group IVelements, and combinations of same.

Formation of nanometer crystals of Group III-V semiconductors isdescribed in copending and commonly assigned Alivisatos et al. U.S. Pat.No. 5,751,018; Alivisatos et al. U.S. Pat. No. 5,505,928; and Alivisatoset al. U.S. Pat. No. 5,262,357, which also describes the formation ofGroup II-VI semiconductor nanocrystals, and which are also assigned tothe assignee of this invention. Also described therein is the control ofthe size of the semiconductor nanocrystals during formation usingcrystal growth terminators. The teachings of Alivisatos et al. U.S. Pat.No. 5,751,018, and Alivisatos et al. U.S. Pat. No. 5,262,357 are eachhereby specifically incorporated by reference.

In one embodiment, the nanocrystals are used in a core/shellconfiguration wherein a first semiconductor nanocrystal forms a coreranging in diameter, for example, from about 20 Å to about 100 Å, with ashell of another semiconductor nanocrystal material grown over the corenanocrystal to a thickness of, for example, 1-10 monolayers inthickness. When, for example, a 1-10 monolayer thick shell of CdS isepitaxially grown over a core of CdSe, there is a dramatic increase inthe room temperature photoluminescence quantum yield. Formation of suchcore/shell nanocrystals is described more fully in a publication by oneof us with others entitled “Epitaxial Growth of Highly LuminescentCdSe/CdS Core/Shell Nanocrystals with Photostability and ElectronicAccessibility”, by Peng, Schlamp, Kadavanich, and Alivisatos, publishedin the Journal of the American Chemical Society, Volume 119, No. 30.1997, at pages 7019-7029, the subject matter of which is herebyspecifically incorporated herein by reference.

The semiconductor nanocrystals used in the invention will have acapability of absorbing radiation over a broad wavelength band. Thiswavelength band includes the range from gamma radiation to microwaveradiation. In addition, these semiconductor nanocrystals will have acapability of emitting radiation within a narrow wavelength band ofabout 40 nm or less, preferably about 20 nm or less, thus permitting thesimultaneous use of a plurality of differently colored semiconductornanocrystal probes with different semiconductor nanocrystals withoutoverlap (or with a small amount of overlap) in wavelengths of emittedlight when exposed to the same energy source. Both the absorption andemission properties of semiconductor nanocrystals may serve asadvantages over dye molecules which have narrow wavelength bands ofabsorption (e.g. about 30-50 nm) and broad wavelength bands of emission(e.g. about 100 nm) and broad tails of emission (e.g. another 100 nm) onthe red side of the spectrum. Both of these properties of dyes impairthe ability to use a plurality of differently colored dyes when exposedto the same energy source.

Furthermore, the frequency or wavelength of the narrow wavelength bandof light emitted from the semiconductor nanocrystal may be furtherselected according to the physical properties, such as size, of thesemiconductor nanocrystal. The wavelength band of light emitted by thesemiconductor nanocrystal, formed using the above embodiment, may bedetermined by either (1) the size of the core, or (2) the size of thecore and the size of the shell, depending on the composition of the coreand shell of the semiconductor nanocrystal. For example, a nanocrystalcomposed of a 3 nm core of CdSe and a 2 nm thick shell of CdS will emita narrow wavelength band of light with a peak intensity wavelength of600 nm. In contrast, a nanocrystal composed of a 3 nm core of CdSe and a2 nm thick shell of ZnS will emit a narrow wavelength band of light witha peak intensity wavelength of 560 nm.

A plurality of alternatives to changing the size of the semiconductornanocrystals in order to selectably manipulate the emission wavelengthof semiconductor nanocrystals exist. These alternatives include: (1)varying the composition of the nanocrystal, and (2) adding a pluralityof shells around the core of the nanocrystal in the form of concentricshells. It should be noted that different wavelengths can also beobtained in multiple shell type semiconductor nanocrystals byrespectively using different semiconductor nanocrystals in differentshells, i.e., by not using the same semiconductor nanocrystal in each ofthe plurality of concentric shells.

Selection of the emission wavelength by varying the composition, oralloy, of the semiconductor nanocrystal is old in the art. As anillustration, when a CdS semiconductor nanocrystal, having an emissionwavelength of 400 nm, may be alloyed with a CdSe semiconductornanocrystal, having an emission wavelength of 530 nm. When a nanocrystalis prepared using an alloy of CdS and CdSe, the wavelength of theemission from a plurality of identically sized nanocrystals may be tunedcontinuously from 400 nm to 530 nm depending on the ratio of S to Sepresent in the nanocrystal. The ability to select from differentemission wavelengths while maintaining the same size of thesemiconductor nanocrystal may be important in applications which requirethe semiconductor nanocrystals to be uniform in size, or for example, anapplication which requires all semiconductor nanocrystals to have verysmall dimensions when used in application with steric restrictions.

c. Affinity Molecules

The particular affinity molecule forming a part of the semiconductornanocrystal probe of the invention will be selected based on itsaffinity for the particular detectable substance whose presence orabsence, for example, in a biological material, is to be ascertained.Basically, the affinity molecule may comprise any molecule capable ofbeing linked to one or more semiconductor nanocrystal compounds which isalso capable of specific recognition of a particular detectablesubstance. In general, any affinity molecule useful in the prior art incombination with a dye molecule to provide specific recognition of adetectable substance will find utility in the formation of thesemiconductor nanocrystal probes of the invention. Such affinitymolecules include, by way of example only, such classes of substances asmonoclonal and polyclonal antibodies, nucleic acids (both monomeric andoligomeric), proteins, polysaccharides, and small molecules such assugars, peptides, drugs, and ligands. Lists of such affinity moleculesare available in the published literature such as, by way of example,the “Handbook of Fluorescent Probes and Research Chemicals”, (sixthedition) by R. P. Haugland, available from Molecular Probes, Inc.

d. The Linking Agents

The semiconductor nanocrystal probe of the invention will usually findutility with respect to the detection of one or more detectablesubstances in organic materials, and in particular to the detection ofone or more detectable substances in biological materials. This requiresthe presence, in the semiconductor nanocrystal probe, of an affinitymolecule or moiety, as described above, which will bond thesemiconductor nanocrystal probe to the detectable substance in theorganic/biological material so that the presence of the detectablematerial may be subsequently ascertained. However, since thesemiconductor nanocrystals are inorganic, they may not bond directly tothe affinity molecule. In this case therefore, there must be some typeof linking agent present in the semiconductor nanocrystal probe which iscapable of linking the inorganic semiconductor nanocrystal to theaffinity molecule in the semiconductor nanocrystal probe. The linkingagent may be in the form of one or more linking agents linking one ormore semiconductor nanocrystals to one or more affinity molecules.Alternatively, two types of linking agents may be utilized. One or moreof the first linking agents may be linked to one or more semiconductornanocrystals and also linked to one or more second linking agents. Theone or more second linking agents may be linked to one or more affinitymolecules and to one or more first linking agents.

One form in which the semiconductor nanocrystal may be linked to anaffinity molecule via a linking agent is by coating the semiconductornanocrystal with a thin layer of glass, such as silica (SiO_(x) wherex=1-2), using a linking agent such as a substituted silane, e.g.,3-mercaptopropyl-trimethoxy silane to link the nanocrystal to the glass.The glass-coated semiconductor nanocrystal may then be further treatedwith a linking agent, e.g., an amine such as3-aminopropyl-trimethoxysilane, which will function to link theglass-coated semiconductor nanocrystal to the affinity molecule. Thatis, the glass-coated semiconductor nanocrystal may then be linked to theaffinity molecule. It is within the contemplation of this invention thatthe original semiconductor nanocrystal compound may also be chemicallymodified after it has been made in order to link effectively to theaffinity molecule. A variety of references summarize the standardclasses of chemistry which may be used to this end, in particular the“Handbook of Fluorescent Probes and Research Chemicals”, (6th edition)by R. P. Haugland, available from Molecular Probes, Inc., and the book“Bioconjugate Techniques”, by Greg Hermanson, available from AcademicPress, New York.

When the semiconductor nanocrystal may be coated with a thin layer ofglass, the glass, by way of example, may comprise a silica glass(SiO_(x) where x=1-2), having a thickness ranging from about 0.5 nm toabout 10 nm, and preferably from about 0.5 nm to about 2 nm.

The semiconductor nanocrystal is coated with the coating of thin glass,such as silica, by first coating the nanocrystals with a surfactant suchas tris-octyl-phosphine oxide, and then dissolving the surfactant-coatednanocrystals in a basic methanol solution of a linking agent, such as3-mercaptopropyl-tri-methoxy silane, followed by partial hydrolysiswhich is followed by addition of a glass-affinity molecule linking agentsuch as amino-propyl trimethoxysilane which will link to the glass andserve to form a link with the affinity molecule.

When the linking agent does not involve the use of a glass coating onthe semiconductor nanocrystal, it may comprise a number of differentmaterials, depending upon the particular affinity molecule, which, inturn, depends upon the type of detectable material being analyzed for.It should also be noted that while an individual linking agent may beused to link to an individual semiconductor nanocrystal, it is alsowithin the contemplation of the invention that more than one linkingagent may bond to the same semiconductor nanocrystal and vice versa; ora plurality of linking agents may be used to link to a plurality ofsemiconductor nanocrystals. In addition, when first and second linkingagents are used, one or more first linking agents may be linked to thesame second linking agent, or more than one second linking agents may belinked to the same first linking agent.

A few examples of the types of linking agents which may be used to linkto both the semiconductor nanocrystal (or to a glass coating on thenanocrystal) and to the affinity molecule in the probe are illustratedin the table below, it being understood that this is not intended to bean exhaustive list:

Linking Agent Structure Name

N-(3-aminopropyl)3- mercapto-benzamide

3-aminopropyl- trimethoxysilane

3-mercaptopropyl- trimethoxysilane

3-(trimethoxysilyl) propylmaleimide

3-(trimethoxysilyl) propylhydrazide

It should be further noted that a plurality of polymerizable linkingagents may be used together to form an encapsulating net or linkagearound an individual nanocrystal (or group of nanocrystals). This is ofparticular interest where the particular linking agent is incapable offorming a strong bond with the nanocrystal. Examples of linking agentscapable of bonding together in such a manner to surround the nanocrystalwith a network of linking agents include, but are not limited to:diacetylenes, styrene-butadienes, vinyl acetates, acrylates,acrylamides, vinyl, styryl, and the aforementioned silicon oxide, boronoxide, phosphorus oxide, silicates, borates and phosphates, as well aspolymerized forms of at least some of the above.

e. Compounds and Probes Having Three-Dimensional Structured LinkingAgents

In one embodiment, the linking agent, including many of those describedabove, may be used in, or as, a three-dimensional structure which may beeither organic or inorganic, and which may be either a solid (porous ornon-porous) or hollow. In the prior art, the use of dye moleculesembedded into latex spheres for diagnostic applications is wellestablished. Perhaps the most common application involves selectivelycoloring the latex sphere using one or more dye molecules and thencoating the sphere with a number of proteins of interest.

The utilization of such a three-dimensional linking agent structure(which may be most easily conceptualized as a sphere) in the compoundand probe of the invention has the added benefit of permitting such alinking agent to have bonded thereto more than one semiconductornanocrystals, as well as one or more affinity molecules (either directlyor through a second linking agent). The three-dimensional linking agentstructure will herein-after be described as a part of a probe(semiconductor nanocrystal, linking agent, and affinity molecule), itbeing understood that the structures described apply to the formation ofa compound (semiconductor nanocrystal and linking agent) as well as aprobe.

The advantage of a compound or probe in which one or more semiconductornanocrystals are bonded to a three-dimensional linking agent structurelies in the ability to simultaneously use a large number ofdistinguishable probes. For example, when using emission of visiblelight as the detectable signal provided by the probe in response toexposure to radiation, multiple distinguishable probes, which eachcontain a single semiconductor nanocrystal of a respectively differentemission color (e.g., blue probe, green probe, red probe) may besimultaneously used. Moreover, a much greater number of distinguishableprobes may be simultaneously used when each probe contains a pluralityof semiconductor nanocrystals, all bound to a single three-dimensionallinking agent in the same probe (e.g., blue-green probe, green-redprobe, blue-red probe, blue-green-red probe). Still further increases incombinations of semiconductor nanocrystals bonded to a three-dimensionallinking agent structure can be achieved by varying the number ofidentically emitting semiconductor nanocrystals bonded to thethree-dimensional linking agent in the same probe in order to providedifferent intensities of detectable signals (e.g. adding a secondblue-emitting semiconductor nanocrystal to a blue-red probe to obtain ablue-blue-red probe, or adding another red-emitting semiconductornanocrystal to a blue-red probe to achieve a blue-red-red probe). Thisfurther increases the total number of probes which can be simultaneouslydistinguished. Similar benefits can be obtained when the detectablesignal or signals provided by the semiconductor nanocrystals in theprobe result from scattering (including diffraction) or absorptionresulting from exposure of the probe to radiation.

Similar to the incorporation of multiple semiconductor nanocrystals in asingle three-dimensionally structured linking agent, multiple affinitymolecules may be linked to the same three-dimensional linking agentstructure to allow a plurality of detectable structures (includingcombinations of detectable structures) to be distinguishably andsimultaneously detected by each semiconductor nanocrystal probe.

In an illustration of the use of multiple affinity molecules in eachsemiconductor nanocrystal probe in testing for Down's syndrome, a subsetof the DNA sequences present on a particular chromosome in the humanbody, such as chromosome 21, may serve as the affinity molecules of asemiconductor nanocrystal probe when attached, in the form of aplurality of separate single stranded DNA fragments, to athree-dimensionally structured linking agent linked to one or more redemitting nanocrystals. A subset of the DNA sequences present on adifferent chromosome, such as chromosome 3, may serve as the singlestranded DNA affinity molecules of another probe when similarly attachedto a different three-dimensionally structured linking agent linked toone or more green emitting nanocrystals. A material comprising a totalDNA sample from a human patient (or an amniocentesis sample), whereinare present one or more detectable substances in the form of singlestranded DNA, may be treated with these semiconductor nanocrystalprobes, resulting in the bonding of the single stranded DNA affinitymolecules of the probes with the single stranded DNA detectablesubstances. This bonding results in the formation of double stranded DNA(in one or both probes), indicative of the presence of one or more DNAsequences (i.e., DNA sequences represented by the single stranded DNAdetectable substances) in the DNA sample. This step may be followed witha step of detecting the bonding of the single stranded DNA affinitymolecules with the single stranded DNA detectable substances by, forexample, adding to the material, which contains the detectablesubstances and has been treated with the semiconductor nanocrystalprobes, a double stranded DNA-binding dye molecule (which may fluoresceblue). The amount of double stranded DNA-binding dye molecules present(determined by amount of blue fluorescence) on a semiconductornanocrystal probe, may be indicative of the amount of double strandedDNA associated with the semiconductor nanocrystal probe. Thus, the bluefluorescence from the probe containing DNA from chromosome 21 indicatesthe bonding of single stranded DNA affinity molecules from chromosome 21with complementary single stranded DNA detectable substances fromchromosome 21, to form double stranded DNA; and the blue fluorescencefrom the probe containing DNA from chromosome 3 indicates the bonding ofsingle stranded DNA affinity molecules from chromosome 3 withcomplementary single stranded DNA detectable substances from chromosome3, to form double stranded DNA.

In this test for Down's Syndrome, the semiconductor nanocrystal probecomprising single stranded DNA affinity molecules from chromosome 3,which emits green light, may serve as a reference probe, wherein theratio of emitted green light to emitted blue light represents thereference amount of double stranded DNA present on a semiconductornanocrystal probe. The semiconductor nanocrystal probe comprising singlestranded DNA affinity molecules from chromosome 21, which emits redlight, may serve as the test probe, wherein the ratio of emitted redlight to emitted blue light (from the test probe) may be compared to theratio of green light to blue light from the reference probe. Adifference between the test and reference ratios may indicate extra orfewer copies of the test chromosome (chromosome 21), in this caseindicating Down s Syndrome. The number of such tests which may besimultaneously performed may be significantly increased by the use of aplurality of colors in each of a plurality of semiconductor nanocrystalprobes.

As stated above, the three-dimensional linking agent structure maycomprise an organic or inorganic structure, and may be a porous ornon-porous solid, or hollow. When the three-dimensional linking agentstructure is a porous (or non-porous) solid the semiconductornanocrystal may be embedded therein, while the semiconductor nanocrystalmay be encapsulated in a hollow three-dimensional linking agentstructure. Whatever the choice of material, it will be appreciated thatwhenever the semiconductor nanocrystal is incorporated into the interiorof the three-dimensional structure of the linking agent, e.g., into a“polymer sphere”, the material comprising the linking agent must both(1) allow a first energy to be transferred from an energy source to theone or more semiconductor nanocrystals (exposing the semiconductornanocrystal to energy), and (2) allow a second energy, provided by theone or more semiconductor nanocrystals in response to exposure to thefirst energy, to be either detected or transferred to a proximalstructure. These transfers of energy may be accomplished by thethree-dimensional linking agent being transparent to the first and/orsecond energies, and/or by the three-dimensional linking agent beingcapable of converting the first and/or second energies to a form whichstill enables the semiconductor nanocrystal probe to either provide adetectable signal or transfer energy to a proximal structure in responseto exposure to energy.

When the three-dimensional linking agent comprises an organic material,the organic material may comprise, for example, one or more resins orpolymers. The semiconductor nanocrystals may be linked to thethree-dimensional linking agent by physically mixing the semiconductornanocrystals with the resin(s) or polymer(s), or may be mixed with themonomer(s) prior to polymerization of the monomer(s) to form thepolymer(s). Alternatively, the semiconductor nanocrystals may be linkedto the three-dimensional linking agent by covalent bonding to either themonomer or the resin or polymer, or the semiconductor nanocrystals maybe linked to the three-dimensional linking agent by adsorption(adherence to the exterior) or absorption (embedded, at least partially,into the interior). Examples of polymers which could be used as organicthree-dimensional linking agents include polyvinyl acetate,styrene-butadiene copolymers, polyacrylates, and styrene-divinylbenzenecopolymers. More than one polymeric chain may be present in thethree-dimensional linking agent, and more than one type of polymer maybe used in the three-dimensional linking agent. The final product couldbe a solid structure, a hollow structure, or a semi-solid porousstructure.

When the three-dimensional linking agent structure comprises aninorganic material, a glass structure such as a glass sphere couldcomprise the transparent structure used to encapsulate one or moresemiconductor nanocrystals therein. The semiconductor nanocrystals couldbe mixed with particles of a low melting point glass, with the mixturethen heated to form the desired three-dimensional structure, e.g., asphere. Alternatively, a porous glass such as a porous silica glasscould be formed into a desired shape (or applied over a solid substrateas a porous coating), followed by incorporation of the semiconductornanocrystals into the pores of the linking agent structure. Thepreviously described glass-coated semiconductor nanocrystals could alsobe modified to provide the three-dimensional linking agent structure ofthis embodiment, for example by providing the glass coating over a coreof such semiconductor nanocrystals or by sintering into athree-dimensional mass a plurality of such glass coated semiconductornanocrystals comprising the same or different semiconductornanocrystals.

An additional increase in the number of three-dimensional structuredprobes which can be distinguishably used may arise from placing one ormore identical semiconductor nanocrystals in one of a plurality ofsubstructures of the three-dimensionally structured probe, andorganizing the various substructures of the probe in such a manner toallow a large number of uniquely identifiable probes to be formed. Forexample, in a single probe, the three-dimensional structured linkingagent may comprise a first semiconductor nanocrystal in a first polymercomprising a first substructure, and a second semiconductor nanocrystalin a second polymer immiscible with the first substructure comprisingsecond substructure.

One example of the arrangement of these substructures is a manneranalogous to the various layers of an onion. In such a construction,different arrangements of several differently emitting semiconductornanocrystals positioned in the various substructure layers may bedistinguished from one another. Therefore, a probe containing an innercore of blue semiconductor nanocrystals, encapsulated by a firstsubstructure layer of red semiconductor nanocrystals, which isencapsulated by a second substructure layer of green semiconductornanocrystals may be distinguished from a probe containing an inner coreof green semiconductor nanocrystals, encapsulated by a firstsubstructure layer of blue semiconductor nanocrystals, which isencapsulated by a second substructure layer of red semiconductornanocrystals. Thus, arranging the different substructures of thesemiconductor nanocrystal probe further increases the number ofdistinguishable probes which may be simultaneously used.

Additionally, various probes whose substructures are assembled indifferent arrangements may be distinguished. For example, a probe whichcomprises red, green and blue semiconductor nanocrystal substructuresordered in an onion-like arrangement may be distinguished from a probewhich comprises red, green, and blue semiconductor nanocrystalsubstructures ordered in a soccer ball-like arrangement.

Therefore, there are a number of different manipulations of thesemiconductor nanocrystals in the probe which results in a very largenumber of distinguishable probes. These manipulations include: varyingthe combinations of different semiconductor nanocrystals in the probe,varying the concentrations of similar and different semiconductornanocrystals in the probe, incorporating semiconductor nanocrystals intoa plurality of substructures in the probe, and varying the arrangementof such substructures containing semiconductor nanocrystals in theprobe.

The incorporation of multiple nanocrystals and/or multiple affinitymolecules into a single probe can be demonstrated in the use of theprobes as the stationary phase in a screen for various nucleic acidsequences, where the nucleic acid sequences in the material beinganalyzed constitute the mobile phase.

A plurality of probes can be prepared which may each comprise a uniquecombination of semiconductor nanocrystals with similar or variedemission wavelengths. Associated with each probe having a uniquesemiconductor nanocrystal combination is a unique combination of one ormore affinity molecules comprising one or more known nucleic acidsequences. In this context, the term “nucleic acid sequence” should beunderstood to include single or double stranded ribonucleic acid (RNA)or deoxyribonucleic acid (DNA) molecules or chemical or isotopicderivatives thereof, each molecule comprising two or more nucleic acidmonomers. A plurality of unidentified nucleic acid sequences comprisingthe detectable substances in a mobile phase material being analyzed maynow be exposed to the above described plurality of probes, e.g. flowedover the stationary phase probes.

The detection, i.e., the identification of the nucleic acid sequences inthe mobile phase bound to the probes involves two aspects. First of allthe occurrence of a bonding event must be ascertained. Secondly theidentification of which probe, and therefore which nucleic acid sequenceor sequences (affinity molecules) of the probe, are bound to the nucleicacid sequences being analyzed. The bonding event itself may bedetermined by detection of a tag (e.g., a dye molecule) which has beenpreviously attached onto all of the nucleic acid sequences beinganalyzed. When bonding occurs, the presence of the tag will correspondspatially to a certain probe or probes. The identification of the typeof nucleic acid sequence or sequences may be determined by the detectionof the signal which corresponds to a unique combination of semiconductornanocrystals within the probe or probes involved in the bonding. Forexample, the probes and material being analyzed may be exposed toradiation of a type which will result in provision of detectable signalsfrom both the dye molecule and the particular probe or probes bonded tothe mobile phase nucleic acid sequences. A spatially identifiable groupof signals from both the dye molecules and semiconductor nanocrystalscan then be detected. The first signal, emanating from the nucleic acidsequences being identified, signifies the presence of a bonded nucleicacid sequence or sequences of any sequence type. The second detectablesignal, emanating from the probe (and the semiconductor nanocrystalstherein), identifies the type of nucleic acid sequence or sequenceswhich are bonded to the probe, by virtue of the known type of nucleicacid sequence or sequences forming the affinity molecule(s) of theprobe.

For example, the material being analyzed and the probes could be exposedto electromagnetic radiation from a laser light source of a frequency atwhich the dye is excitable and which will also excite the semiconductornanocrystals in the probe. The resulting detectable signals from the dyemolecules and the probe or probes, could be visible light emissions ofone or more frequencies signifying the presence of bonded nucleic acidsequences (the light from the dye molecules) and the identity of theparticular probe bonded thereto (the light from the semiconductornanocrystals in the probe). When the spatial locations of both the dyemolecule emission and the probe emission correspond, this would signifythe presence of particular nucleic acid sequences bonded to particularprobes known to emit light of the detected frequencies.

Thus, once bonded to one or more nucleic acid sequences from the mobilephase being analyzed, a plurality of similar or different probes maythen be identified according to the unique combination of semiconductornanocrystals present in each probe. The probes may be identified eitherone after the other or simultaneously. The identification of each probethen allows the identification of the unique nucleic acid sequence orcombination of nucleic acid sequences bound to the probe by way of theknown nucleic acid sequence comprising the affinity molecule on thesurface of each probe. In this way, a large number of different nucleicacid sequences may be rapidly screened and identified.

It should be noted that while it is contemplated that each affinitymolecule comprising a unique, known nucleic acid sequence or sequenceswill be specifically bonded to a particular unidentified nucleic acidsequence or sequences being analyzed for, thus making identificationprecise, other uses may be contemplated. For example, a probe could bedesigned having, as its affinity molecule portion, a particular knownnucleic acid sequence or sequences which would be bondable to an entiregroup of related unidentified nucleic acid sequences, thus permittinguse of the probe as a broad identification or screening agent.

f. Exposure of the Probe to Energy and Detection ofEmission/Absorption/Scattering

Upon exposure of the semiconductor nanocrystal probe to energy, some orall of the energy may be transferred to the semiconductor nanocrystalprobe. In response to exposure to this energy, the semiconductornanocrystal probe may provide a plurality of detectable signals. Thesedetectable signals include (1) emission of electromagnetic radiation,(2) absorption of radiation, and (3) scattering, including diffraction,of radiation.

The emission properties of the semiconductor nanocrystal probe may bevery useful in a plurality of applications. As previously mentioned, thesemiconductor nanocrystal probe of the invention is capable of beingexcited over a broad bandwidth, yet exhibits emission in a narrowwavelength band, in contrast to the dye molecules used in the prior art.Thus electromagnetic radiation of wavelength ranging from x-ray toultraviolet to visible to infrared waves may be used to excite thesemiconductor nanocrystals in the probe. In addition, the semiconductornanocrystals are capable of excitation from bombardment with a particlebeam such as an electron beam (e-beam). Furthermore, because of thebroad bandwidth at which the semiconductor nanocrystals are excitable,one may use a common excitation source for the simultaneous excitationof several probes, e.g., several probes which give off radiation atdifferent frequencies, thus permitting simultaneous excitation anddetection of the presence of several probes indicating, for example, thepresence of several detectable substances in the material beingexamined.

Thus, for example, a laser radiation source of a given frequency, e.g.,blue light, may be used to excite a first semiconductor nanocrystalprobe capable of emitting radiation of a second frequency, e.g., redlight, indicating the presence, in the material being illuminated, of afirst detectable substance to which the particular red light-emittingsemiconductor nanocrystal probe has bonded. At the same time, the sameblue light laser source may also be exciting a second semiconductornanocrystal probe (in the same material) capable of emitting radiationof a third frequency, e.g., green light, indicating the presence, in thematerial being illuminated, of a second detectable substance to whichthe particular green light-emitting semiconductor nanocrystal probe hasbonded. Thus, unlike the prior art, multiple excitation sources need notbe used (because of the broad bandwidth in which the semiconductornanocrystal probe of the invention is capable of being excited), and thenarrow band of emission of the specific semiconductor nanocrystals ineach probe makes possible the elimination of sequencing and/or elaboratefiltering to detect the emitted radiation.

Another detectable signal provided by a semiconductor nanocrystal probein response to radiation is absorption. The presence of thesemiconductor nanocrystal probe, bound to a detectable substance in abiological material, may be indicated by detection of absorption ofradiation by the semiconductor nanocrystal probe. Since thesemiconductor nanocrystal probe has such a wide wavelength band ofabsorption, detection of the semiconductor nanocrystal probe may becarried out over a wide range of wavelengths, according to therequirements of the detection process. For example, many biologicalmaterials strongly absorb visible and ultraviolet radiation, but do notstrongly absorb x-ray radiation. Such a biological material containing adetectable substance may be treated with a semiconductor nanocrystalprobe. Presence of the semiconductor nanocrystal probe bonded with thedetectable substance may then be indicated by detection of an absorptionof x-rays.

The semiconductor nanocrystal probe of the invention may also provide adetectable scattering signal in response to exposure to energy. Thisdetectable scattering signal may be a diffraction signal. Thus, forexample, presence of a detectable substance within a material treatedwith a semiconductor nanocrystal probe (wherein the semiconductornanocrystal probe is bonded to the detectable substance) may beindicated by the detection of a change in the scattering cross sectionor in diffraction of radiation upon exposure of the material toradiation.

The semiconductor nanocrystal probe of the invention may also be used insuch a way that, instead of providing a detectable signal in response toradiation, it transfers energy to a proximal structure. This proximalstructure, in response to the energy transfer, may then (1) provide adetectable signal, (2) undergo chemical or conformational changes, (3)transfer energy to a second proximal structure, or (4) any combinationthereof. This may be achieved by introducing the semiconductornanocrystals and the material together by any of the above methods, andthen exposing the material to energy. It should be noted that a proximalsource may be used to transfer energy from the proximal source to theprobe (as will be described below) in contrast to the aforesaid transferof energy from the probe to a proximal structure.

g. General Use of the Probe

In general, the probe may be used in treating a material to determinethe presence of a detectable substance by introducing the probe, forexample, dispersed in a suitable carrier such as an aqueous solution(e.g., an saline solution), into the material to permit the affinitymolecule of the probe to bond to the detectable substance (if suchdetectable substance is present in the material). After introduction ofthe probe into the material, unbonded probes may be optionally removedfrom the material, leaving only bonded probes. In either event, thematerial (and probes therein) may be exposed to an energy source capableof causing the probe(s) to provide a detectable signal. When theunbonded probes have not been removed, presence of the bonded probes canbe determined (and distinguished from the unbonded probes) by aplurality of methods, including determining the spatial segregation ofmore intense detectable signals arising as a result of the localizationof the bonded probes, as opposed to random dispersion (resulting inspatially random detectable signals) of the unbonded semiconductornanocrystal probes.

As an alternative to adding the semiconductor nanocrystal probe to thematerial, the material may be in a carrier, such as an aqueous solution,and this material may be introduced into a compartment containing thesemiconductor nanocrystal probe. The semiconductor nanocrystal probe mayitself be in a carrier, or may be attached to a solid support. Presenceof the detectable substance within the material may be determined by anymethod which is capable of indicating the bonding of the affinitymolecule of the probe to the detectable substance. This may beaccomplished, for example, by separating components of the material andexposing the components of the material to radiation, wherein asemiconductor nanocrystal probe, if present, may provide a detectablesignal in response to exposure to radiation.

The carrier mentioned above is any type of matter that has little or noreactivity with the semiconductor nanocrystal probe, and enables storageand application of the semiconductor nanocrystal probe to the materialto be treated. Such a material will often be a liquid, including manytypes of aqueous solutions, including biologically derived aqueoussolutions (e.g. plasma from blood). Other liquids include alcohols,amines, and any other liquid which neither reacts with nor causes thedissociation of the components of the semiconductor nanocrystal probe.The carrier also comprises a substance which will not interfere with thetreatment or analysis being carried out by the probe in connection withthe detectable substance in the material.

A further use of the semiconductor nanocrystal probe of the invention isto provide a detectable signal in response to energy transferred fromone or more spatially proximal sources. In this context, “energytransfer” is meant the transfer of energy from one atom, molecule, orany other substance (e.g. a polymer, a gel, a lipid bilayer, etc.) toanother atom, molecule, or any other substance by either (1) a radiativepathway (e.g., emission of radiation by a first atom or moleculefollowed by scattering—including diffraction—and/or absorption of theemitted radiation by a second atom or molecule); or (2) a non-radiativepathway (e.g., fluorescence resonance energy transfer, or FRET, from afirst atom or molecule to a second atom or molecule). By use of the term“proximal source” is meant an atom, a molecule, or any other substancewhich is capable of transferring energy to and/or receiving energytransferred from another atom or molecule or any other substance. By useof the term spatially proximal source is meant a proximal source spacedsufficiently close to enable energy to be transferred from a proximalsource to a semiconductor nanocrystal probe. For example, in the case ofFRET, a spatially proximal source comprises a proximal source spaced 10nm or less from the semiconductor nanocrystal probe. In the case of thetransfer of radioactive energy, a spatially proximal source comprises aproximal source spaced 1 μm or less from the semiconductor nanocrystalprobe.

The energy transferred from a proximal source to the semiconductornanocrystal probe may originate from the proximal source (e.g.,radioactive decay of an atom or atoms within the proximal source) or mayarise as a result of excitation by an energy source separate from theproximal source (e.g., excitation of a proximal source dye molecule by alaser) as will be explained below. An illustration of a radiativepathway of energy transfer is the transfer of gamma radiation from aradioactive nucleus (of the proximal source) to a semiconductornanocrystal probe. The transferred gamma radiation may then be absorbedby the semiconductor nanocrystal probe, which, in response to absorptionof the gamma radiation, provides a detectable emission signal ofelectromagnetic radiation. An illustration of a non-radiative pathway isactivation of the semiconductor nanocrystal by a FRET from a proximalsource which has been externally excited, as will be described below.

Such a spatially proximal energy transfer may be useful in measuring theconcentration of the proximal source, as well as the distance of theproximal source from the probe. Spatially proximal energy transfer canalso be used in the detection of an event which causes the source fromwhich energy is transferred to become spatially proximal to the probe.

One illustration of a spatially proximal energy transfer using asemiconductor nanocrystal probe is as a concentration indicator, whereinthe semiconductor nanocrystal probe, in essence, acts as an energytransfer reporter. That is, the semiconductor nanocrystal probe, forexample, may provide a detectable emission signal, the strength of whichis a function of the local concentration of proximal sources from whichthe energy is transferred. This permits the probe to be used todetermine the concentration of proximal sources from which energy istransferred. A possible application of this method would be to measurethe amount of a zinc finger protein, such as the RAG1 protein,synthesized by a cell during a specific length of time using apulse-chase experiment. The cell mixture may be pulsed with an additionof radioactive zinc ions to the growth medium and may, after a specificlength of time, be chased by addition of non-radioactive zinc ions inlarge excess (e.g., greater than 100-fold) of the radioactive zinc ions.Such a pulse-chase experiment will result in one or more radioactivezinc ions incorporated only in zinc containing proteins synthesizedduring the specified length of time between the pulse and the chase. Thecells may then be lysed to yield a soluble cell extract comprising oneor more zinc containing proteins. A semiconductor nanocrystal probecomprising an affinity molecule, such as an antibody, which selectivelybonds to a particular zinc finger protein may then be added to thesoluble cell extract, allowing the semiconductor nanocrystal probe tobond to the particular zinc finger protein. The concentration of theparticular zinc finger protein, comprising one or more radioactive zincions, and acting as the proximal source from which energy istransferred, bonded to semiconductor nanocrystal probe may be indicatedby a detectable signal provided by the semiconductor nanocrystal probein response to energy transferred from the radioactive zinc ion of thebonded particular zinc finger protein.

Another illustration of a spatially proximal energy transfer using thesemiconductor nanocrystal probe is as a distance indicator. The strengthof the detectable signal, for example, an emission, from a semiconductornanocrystal probe is a function of the distance (provided that thedistance is less than about 1 μm) between the semiconductor nanocrystalprobe and the proximal source from which energy is transferred.Therefore, the detectable signal provided by the semiconductornanocrystal probe may serve as an indicator of the distance between thesemiconductor nanocrystal probe and the proximal source from whichenergy is transferred. A possible application for this is in the abilityto determine spatial proximity of individual subunits of a multi-subunitcomplex within a cell, such as a transcriptional initiation complex, aribosome, a lipid-lipoprotein complex, etc. For example, a semiconductornanocrystal probe may bond with a protein subunit of a ribosome, while aRNA subunit of the ribosome may be labeled with a radioactivephosphorous atom, which serves as the proximal source from which energyis transferred (in this illustration, the energy transferred from theproximal source to the semiconductor nanocrystal probe originates fromthe proximal source). Since the strength of the emission of a detectablesignal is a function of the distance between the semiconductornanocrystal probe and the proximal source from which energy istransferred, the signal provided by the semiconductor nanocrystal probebonded to the protein subunit indicates the approximate distance betweenthe portion of the protein subunit bonded to the semiconductornanocrystal probe and the portion of the RNA which contains theradioactive phosphorus atom from which the energy is transferred.

The spatially proximal energy transfer use of the semiconductornanocrystal probe also may be utilized to detect the occurrence of anevent. This event, for example, may cause the source from which energyis transferred to be located spatially proximal to the semiconductornanocrystal probe. Since the detectable signal is a function of thedistance between the proximal source from which energy is transferredand the semiconductor nanocrystal probe, the signal provided by thesemiconductor nanocrystal probe may yield information reflective of anevent which causes the source to be sufficiently proximal (less thanabout 10 nm) to enable energy to be transferred from the proximal sourceto the semiconductor nanocrystal probe. By way of illustration, asemiconductor nanocrystal probe may bond with a thyroid hormone receptormolecule. A thyroid hormone such as thyroxine may be labeled with aradioactive iodine atom, which serves as the source from which energy istransferred. An event which causes the thyroxine to bond to the thyroidhormone receptor will also cause the radioactive iodine atom in thethyroxine to be spatially proximal to the semiconductor nanocrystalprobe. Therefore, this bonding event will cause energy to be transferredfrom the radioactive iodine atom to the semiconductor nanocrystal probewhich may provide a detectable signal in response to the energytransfer. The detectable response will thus serve as an indicator of theevent of thyroxine bonding to the thyroid hormone receptor.

The energy transferred from one or more proximal sources to one or moresemiconductor nanocrystal probes may either originate from the proximalsource (as in the example of radioactive decay of an atom or atomswithin the proximal source), or may arise as a result of excitation ofthe one or more proximal sources by an energy source separate from theproximal sources. By use of the term energy source separate from theproximal source is meant any source of radiation or any other energywhich transfers energy to the proximal source. The energy sourceseparate from the one or more proximal sources may either be spatiallydistant or spatially proximal to the proximal source from which energyis transferred to the semiconductor nanocrystal probe. Thus, the energymay be transferred from a spatially distant energy source such as, forexample, a laser or particle beam; or the energy may be transferred froma second spatially proximal source from which second proximal source theenergy transferred may either originate, or arise as a result ofexcitation by an energy source separate from the second proximal source.For example, a laser beam may be used to excite a second proximalsource, the second proximal source then excites the first proximalsource, and the first proximal source excites the semiconductornanocrystal probe; or a second proximal source may be a radioactive atomwhich excites the first proximal source which excites the semiconductornanocrystal probe. It will be understood that more than two proximalenergy sources can be utilized to transfer energy in a cascading effect.Included in pathways of excitation of the proximal source by a separatesource is the case where the separate source is a particle beam which,when the proximal source is exposed to the particle beam, may cause anuclear event in the proximal source. The proximal source may thentransfer energy to the semiconductor nanocrystal probe as a result ofthe nuclear event caused by exposure of the proximal source to theparticle beam.

When the excitation of the proximal source arises as a result of energytransferred from a separate energy source (e.g., a laser beam) theenergy transfer from the proximal source to the semiconductornanocrystal probe may be accomplished by FRET, as previously mentioned.Thus, an energy source separate from the proximal source, such as alaser, may excite a proximal source. The proximal source, as a result ofrelaxing from an excited state, may transfer energy via fluorescenceresonance energy transfer to the semiconductor nanocrystal probe whenthe proximal source is less than about 10 nm from the semiconductornanocrystal probe. The semiconductor nanocrystal probe may then providea detectable signal such as electromagnetic radiation in response to theenergy transfer from the proximal molecule. An illustration of both theexcitation of the proximal molecule by an energy source separate fromthe proximal energy source and the use of FRET as the pathway of energytransfer from the proximal source to the probe may be derived from thepreviously described ribosomal example. In contrast to the previousexample which used an RNA subunit of the ribosome labeled with aradioactive phosphorus atom as the proximal source, a dye molecule maybe attached to the RNA subunit instead of the radioactive phosphorousatom. The proximal source RNA subunit with attached dye molecule maythen be excited by a separate source, for example a laser beam. Theexcited proximal source RNA subunit may transfer energy to asemiconductor nanocrystal probe by way of a non-radiative energytransfer pathway such as FRET, which may provide a detectable signal inresponse to the energy transferred from the proximal source RNA subunit.

The use of a proximal source to transfer energy to a semiconductornanocrystal probe may be modified in such a way as to enable a proximalsource to transfer energy to a plurality of semiconductor nanocrystalprobes. By way of illustration, in the previous example using an RNAmolecule labeled with a dye molecule as the proximal molecule, aplurality of RNA proteins may be labeled, each with a differentlyemitting semiconductor nanocrystal probe. Fluorescence resonance energymay be transferred from the dye molecule to one or more of thedifferently emitting semiconductor nanocrystal probes. The detectablesignals provided by the one or more differently emitting semiconductornanocrystal probes may then signify proximity between the dye and theone or more semiconductor nanocrystal probes.

Since semiconductor nanocrystals of specific wavelength emission may beselected for use in a particular probe, a semiconductor nanocrystalprobe may be exposed to, for example, a radioactive atom emitting gammaradiation from a proximal source, and the wavelength of the emissionfrom the semiconductor nanocrystal probe, in response to exposure togamma radiation from the proximal source, may be selected to beultraviolet radiation, according to the nature of the semiconductornanocrystal within the semiconductor nanocrystal probe. Alternatively,the wavelength of the emission of the semiconductor nanocrystal inresponse to exposure to, for example, gamma radiation from the proximalsource may be selected to be red light. The ability to provide multipleand selectable different emissions in response to exposure to theidentical radiation allows a plurality of differently emittingsemiconductor nanocrystal probes to be used simultaneously. Thesimultaneous use of a plurality of probes which each emit differentwavelengths of electromagnetic radiation can be used, for example, in aconfiguration where proximity between a specific semiconductornanocrystal probe and a source from which energy is transferred to thesemiconductor nanocrystal probe may be determined by the specificwavelength of the emission from the semiconductor nanocrystal probe. Forexample, three semiconductor nanocrystal probes which differ in thevisible light they emit (e.g., blue, green, and red emittingsemiconductor nanocrystal probes) could be attached to portions of anassociation of molecules (e.g., an organelle). Presence of a certainmolecule with a radioactive atom attached (therefore acting as theproximal source) in proximity to one specific semiconductor nanocrystalprobe results in emission of a specific color, indicating proximitybetween the certain molecule and the specific semiconductor nanocrystalprobe and its associated affinity molecule.

Similar to the use of multiple semiconductor nanocrystals, it ispossible to use multiple proximal sources capable of transferring energyto one or more semiconductor nanocrystal probes.

Similar to the process in which energy is transferred from one or moreproximal sources to one or more semiconductor nanocrystal probes, energymay also be transferred from one or more semiconductor nanocrystalprobes to one or more proximal structures in response to exposure of thesemiconductor nanocrystal probe to energy. The term “proximal structure”as used herein may be an atom, a molecule, or any other substance (e.g.a polymer, a gel, a lipid bilayer, and any substance bonded directly toa semiconductor nanocrystal probe) which is capable of receiving energytransferred from another atom or molecule or other substance (includinga semiconductor nanocrystal probe). The proximal structure, in responseto the energy transferred from the semiconductor nanocrystal probe, may(1) provide a detectable signal, (2) undergo chemical and/orconformational changes, (3) transfer energy to one or more secondproximal structures, or (4) any combination thereof. As used herein, a“second proximal structure” is a proximal structure to which energy istransferred from a first proximal structure which has received energyfrom a semiconductor nanocrystal probe. The second proximal structure,in response to the energy transferred from the first proximal structuremay (1) provide a detectable signal, (2) undergo chemical and/orconformational changes, (3) transfer energy to one or more thirdproximal structures (where a “third proximal structure” is one to whichenergy has been transferred from a second proximal structure), or (4)any combination thereof. It will be understood that the transfer ofenergy between proximal structures may be further extended beyond athird proximal structure in a cascading effect.

An illustration of the use of a semiconductor nanocrystal probe totransfer energy to a proximal structure which provides a detectablesignal is as follows. A semiconductor nanocrystal probe may be used toprovide an emission of a narrow wavelength band in the blue region ofvisible light in response to excitation over a broad wavelength band ofradiation. When this semiconductor nanocrystal probe is spatiallyproximal to a dye molecule (the dye molecule herein is acting as theproximal structure), the dye molecule may then become excited upontransfer of energy from the semiconductor nanocrystal probe. The exciteddye molecule may then be capable of providing a detectable red lightemission in response to excitation by the energy transfer from thesemiconductor nanocrystal.

An illustration of the use of a semiconductor nanocrystal probe totransfer energy to a proximal structure which, in response to the energytransferred from the semiconductor nanocrystal probe, undergoes chemicalchanges, is the use of semiconductor nanocrystals to break covalentbonds. A semiconductor nanocrystal probe may be exposed to energy, andmay then transfer energy to a proximal structure in response to theexposure to energy. The energy transferred may be, for example,electromagnetic radiation which is capable of inducing a photolyticcleavage (or photolysis) of a covalent bond in a proximal structure.This action of photolysis may also result in the detachment of a portionof the proximal structure. This detached portion of the proximalstructure may be, for example, a molecule used for therapeutic purposessuch as a molecule with cytotoxic properties. This use of thesemiconductor nanocrystal probe to break covalent bonds may becontrolled in a dosage specific manner, according to the extent ofexposure of the semiconductor nanocrystal probe to radiation. Thiscontrol of the exposure of the semiconductor nanocrystal probe toradiation may result in control of the energy transferred to theproximal structure, which controls the photolytic cleavage of thecovalent bond, and ultimately controls the detachment of the portion ofthe proximal structure. Additionally, the portion of the proximalstructure may be detached in a spatially specific manner, according tothe specificity of the one or more affinity molecules of thesemiconductor nanocrystal probe.

This use of the semiconductor nanocrystal probe to break covalent bondsin the proximal structure may be particularly effective when the energytransferred to the semiconductor nanocrystal probe has a long wavelengthwhich is transparent to the material surrounding the semiconductornanocrystal probe. For example, a semiconductor nanocrystal probe may beexposed to electromagnetic radiation from a laser which emits at awavelength of 700 nm (infrared radiation). Materials such as biologicalmaterials absorb very little radiation at 700 nm, but a semiconductornanocrystal probe may absorb radiation at 700 nm It is common forphotolytic cleavages to require ultraviolet radiation for activation. Anadvantage of the semiconductor nanocrystal probe of the invention isthat it may be made to transfer energy corresponding to ultravioletradiation when exposed to infrared radiation as a result of a processtermed two-photon absorption. Two-photon absorption may occur when asemiconductor nanocrystal probe is exposed to radiation in such a waythat it simultaneously absorbs two quanta of radiation (i.e., twophotons), and the resultant level of excitation of the semiconductornanocrystal probe is twice as large as the level of excitation thesemiconductor nanocrystal probe would have if it had absorbed a singlequantum of radiation. By the physical relationship between energy andwavelength of radiation (E=hc/λ, where E is energy, h and c areconstants, and λ is wavelength), a level of excitation, corresponding totwo quanta of a first type of radiation with a certain wavelength, wouldcorrespond to the level of excitation caused by absorption of a singlequantum of a second type of radiation with a wavelength half that of thefirst type of radiation. Thus, if a semiconductor nanocrystal probesimultaneously absorbs two photons with wavelength of 700 nm, theexcitation level of the semiconductor nanocrystal probe will be the sameas the excitation level of a semiconductor nanocrystal probe whichabsorbs a single photon with a wavelength of about 350 nm (ultravioletradiation). A semiconductor nanocrystal probe which has been excited bytwo-photon absorption may thus transfer energy, for example, by emittingelectromagnetic radiation with a shorter wavelength than the wavelengthof the radiation to which the semiconductor nanocrystal probe wasexposed.

As an illustration of the use of this two-photon absorption, asemiconductor nanocrystal probe, comprising one or more affinitymolecules which may specifically bond to one or more detectablesubstances representative of the presence of a cancerous cell or tissue,may be exposed to radiation from an infrared laser emitting at 700 nm.This semiconductor nanocrystal probe may then be excited by the infraredradiation (through the process of two-photon absorption), and may thenemit ultraviolet radiation (which has a shorter wavelength—e.g. about350 nm). This emitted radiation in the ultraviolet range (or energytransferred by some other process, such as by FRET) may then cause aphotolytic cleavage in a proximal structure, which results in acytotoxic molecule being detached from the proximal structure and actingas a toxin to the cancerous cell or tissue.

Another illustration of the response to the energy transferred from thesemiconductor nanocrystal probe to the proximal structure resulting inthe proximal structure undergoing chemical or conformational changes mayresult when the energy transferred from the semiconductor nanocrystalprobe to the proximal structure is heat energy. This transfer of heatenergy may result in a conformational change such as the heat-induceddenaturation of a protein. A semiconductor nanocrystal probe may be ableto absorb radiation which is not absorbed by the material surroundingthe semiconductor nanocrystal probe. In response to exposure of thesemiconductor nanocrystal probe to radiation, the semiconductornanocrystal probe may transfer heat energy to a proximal structure,resulting in a local heating of structures proximal to the semiconductornanocrystal probe. In response to this local heating, the proximalstructure may (1) undergo a chemical or conformational change, and/or(2) transfer energy to a second proximal structure. Thus, exposure of amaterial to radiation (to which radiation the material is transparent)may result in local heating within the material. The heat energytransferred from the semiconductor nanocrystal to the proximal structuremay then result in chemical or conformational changes in the proximalstructure, and/or some or all of the heat energy may be transferred to asecond proximal structure which itself could undergo chemical orconformational changes and/or transfer some or all of the heat energy toa third proximal structure, and so on. As in the example of thephotolytically detached cytotoxic molecule, use of the semiconductornanocrystal probe to cause transfer of heat energy may be controlled ina dosage specific manner, according to the extent of exposure of thesemiconductor nanocrystal probe to radiation. Additionally, the heatenergy may be transferred in a spatially specific manner, according tothe specificity of the one or more affinity molecules of thesemiconductor nanocrystal probe.

The amount of heat energy transferred to a proximal structure from asemiconductor nanocrystal probe in response to exposure to radiation maybe enough to generate a large amount of local heating due to the highdegree of stability and the large extinction coefficients characteristicof nanocrystals. In a specific example of the extent of local heatingwhich may occur, when semiconductor nanocrystals (which emit infraredradiation) are present in a tissue at a concentration of about 0.0001grams of semiconductor nanocrystals per gram of tissue, and thesenanocrystals are exposed to an ultraviolet excitation source (or a twophoton absorption source capable of exciting with an ultravioletexcitation energy), the heat energy transferred by these semiconductornanocrystals over 1,000,000 photocycles (about one second of exposure toa saturating laser) in response to exposure to radiation may cause thetissue to increase in temperature by about 25 C. This large amount oflocal heating may be, for example, great enough to kill local cells andtissue; and therefore this use of the semiconductor nanocrystal probe totransfer heat energy may be applied to the treatment of cancerous cellsor other nefarious cells and tissues.

Energy transfer from one or more semiconductor nanocrystal probes to oneor more proximal structures may take place in a manner similar to any ofthe previously described transfers of energy from one or more proximalsources to one or more semiconductor nanocrystal probes. Therefore, asemiconductor nanocrystal probe may transfer energy to a proximalstructure by way of radiative or non-radiative (e.g., FRET) pathways.The energy transferred from a semiconductor nanocrystal probe to aproximal structure by a radiative pathway may include particle andelectromagnetic radiation. The energy transfer from a semiconductornanocrystal probe to a proximal structure may occur as a result ofenergy transferred from an energy source separate from the semiconductornanocrystal probe. This energy source separate from the semiconductornanocrystal probe may either be a spatially distant energy source suchas, for example, a laser or particle beam; or the energy may betransferred from a spatially proximal source, as previously discussed.This includes, for example, a spatially distant energy source which maytransfer energy to a spatially proximal source, which may transferenergy to a semiconductor nanocrystal probe, which may transfer energyto a proximal structure.

Prior to using a semiconductor nanocrystal probe in a process comprisingexposure of the semiconductor nanocrystal probe to energy, thesemiconductor nanocrystal probe may be used as a precursor which may besubjected to further synthetic steps. These further synthetic steps mayresult in formation of a modified semiconductor nanocrystal probe whichhas a different affinity molecule than the affinity molecule of theprecursor semiconductor nanocrystal probe. For example, a semiconductornanocrystal probe, having one or more nucleic acid monomers as itsaffinity molecule portion, may serve as a precursor (primer) in aprocess for synthesizing DNA in large amounts, such as polymerase chainreaction (PCR); and the final PCR product may be a modifiedsemiconductor nanocrystal probe having an affinity molecule with agreater number of nucleic acid monomers than the affinity molecule ofthe precursor semiconductor nanocrystal probe. The synthetic steps towhich the semiconductor nanocrystal probe may be subjected include, forexample, any method of nucleic acid synthesis (by use of the term,nucleic acid synthesis it is meant any enzymatic process of synthesizingnucleic acid strands using nucleic acid monomers). In any such nucleicacid synthesis (including the above PCR case), the precursorsemiconductor nanocrystal probe is understood to comprise one or morenucleic acid strands, each comprising a number of nucleic acid monomerssufficient to allow the precursor semiconductor nanocrystal to be usedas a primer in a nucleic acid synthesis reaction such as PCR (thenucleic acid strands often having from 1 to about 50 nucleic acidmonomers) as the one or more affinity molecules portion of thesemiconductor nanocrystal probe. The term “nucleic acid strand” shouldbe understood to include a plurality of single or double strandedribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules orchemical or isotopic derivatives thereof, each molecule comprising twoor more nucleic acid monomers. This nucleic acid strand affinitymolecule portion may be modified by extending the nucleic acid strandsby addition of nucleic acid monomers according to the desired sequenceof the nucleic acid synthesis (chains may vary in length from 1 morenucleic acid monomer than the precursor, or primer, to as much as500,000 nucleic acid monomers, or more if desired). This modifiedsemiconductor nanocrystal probe is understood to have all of theproperties and potential uses of any semiconductor nanocrystal probe.That is, the modified semiconductor nanocrystal probe is capable ofbonding with one or more detectable substances, and is capable ofproviding a detectable signal in response to exposure to energy. Thismay include, for example, use of the modified semiconductor nanocrystalprobe (comprising an affinity molecule with a modified DNA sequence) asa fluorescent marker in a plurality of nucleic acid based assays,including DNA sequencing assays and hybridization assays such asfluorescence in-situ hybridization and comparative genomichybridization.

Another advantage of the semiconductor nanocrystal probe (or asemiconductor nanocrystal compound) of the invention is in any processwhich involves elevated temperatures. As used herein, “elevatedtemperatures” are understood to include temperatures from roomtemperature (about 25 C) up to the temperature at which the particularsemiconductor nanocrystal probe undergoes thermal degradation. Typicallythis may occur at temperatures of about 150 C or even as low as 100 C.Because of the high degree of thermal stability of the semiconductornanocrystals, semiconductor nanocrystal probes (or semiconductornanocrystal compounds) may withstand use at elevated temperatures,including use in processes which comprise thermal cycling steps (i.e.,processes which comprise one or more steps in which the temperature iscycled between a low temperature and a high temperature, such as theaforementioned PCR). For example, as discussed above, a precursorsemiconductor nanocrystal probe may be used in PCR, which requiresmultiple steps in which the temperature is cycled between a lowtemperature (the DNA synthesis step) and a high temperature (the DNAstrand separation step). The high temperature of the PCR reactionmixture may be about 95₁ C, a temperature at which many dye moleculesdegrade. The thermal stability properties of the semiconductornanocrystal probe enable it to withstand the thermal cycling of PCR.

In addition to the use of semiconductor nanocrystal probes in PCR, theadvantage of the high degree of thermal stability of the semiconductornanocrystal probes may be applied to any other processes which mayrequire elevated temperatures, such as use in heat shock methods, ormethods using thermostable organisms or biomolecules derived fromthermostable organisms.

An illustration of the simultaneous use of a plurality of differentsemiconductor nanocrystal probes is when a plurality of semiconductornanocrystal probes are used in flow cytometry analysis. Flow cytometry,as used in the prior art, involves contacting a material, containingcells, with one or more dyes, or dye conjugated affinity molecules,which are capable of detecting certain molecules or substances on thesurface or interior of those cells. The presence of the dye molecules onthe surface or interior of a cell (and, hence, the presence of thecertain molecule with which the dye interacts) is detected by flowingthe material through a compartment which is transparent to both theenergy to which the material is exposed, and to the detectable signalprovided by the dye in response to exposure to energy. As the cells arewithin the transparent compartment, the cells are exposed to energy,such as electromagnetic radiation, which is capable of being absorbed bythe dye. The dye, as a result of exposure to the electromagneticradiation, emits a detectable signal, such as electromagnetic radiationof a different wavelength than that to which the material is exposed.When a plurality of dyes are used to indicate the presence of aplurality of substances on the surface or interior of the cells, thematerial containing the cells may be flowed through a plurality oftransparent compartments, and the presence of a plurality of differentdyes may be tested one at a time (i.e. consecutively) or a few at a time(maximum of three simultaneous detections).

In accordance with the invention, instead of using a dye molecule, amaterial containing cells may alternatively be contacted with asemiconductor nanocrystal probe (actually a plurality of probe, but allproviding the same detectable signal in response to energy). Thesemiconductor nanocrystal probe may bond to one or more detectablesubstances, if any are present, on the surface or interior of the cells,to which the affinity molecules of the semiconductor nanocrystal probeare capable of bonding. Detection of the presence of the semiconductornanocrystal probe (and hence, the presence of one or more specificdetectable substances to which the semiconductor nanocrystal probe isbonded) may take place by first contacting the material containing thecells with the semiconductor nanocrystal probe. The material is thenflowed through a transparent compartment wherein the material is exposedto energy such as, for example, ultraviolet laser radiation. Thepresence of the semiconductor nanocrystal probe may be indicated by adetectable signal such as, for example, emission of red light, providedby the semiconductor nanocrystal probe in response to exposure toenergy. Detection of the detectable signal provided by the semiconductornanocrystal probe, therefore, may indicate the presence of one or moredetectable substances, on the surface or interior of cells, to which thesemiconductor nanocrystal probe is bonded.

Use of a plurality of groups of semiconductor nanocrystal probes (eachof which groups provide the same detectable signal in response toexposure to energy) may be conducted in a manner similar to the aboveuse of a single semiconductor nanocrystal probe. The material containingthe cells may be contacted with a plurality of semiconductor nanocrystalprobes, and the material is then flowed through a plurality oftransparent compartments. In each compartment, the presence of aspecific semiconductor nanocrystal probe bonded to one or moredetectable substances may be indicated by a particular detectable signalprovided by the specific semiconductor nanocrystal probe. However,unlike the prior art, since each separate semiconductor nanocrystalprobe is capable of producing a detectable signal (in response toenergy) which is distinguishable from the detectable signals produced byother semiconductor nanocrystal probes which have been exposed to thesame energy, the presence of more than one semiconductor nanocrystalprobe, each bonded to one or more different detectable substances, maybe simultaneously detected in a single compartment.

Furthermore, methods of using one or more semiconductor nanocrystalprobes to detect one or more detectable substances on the surface orinterior of cells may not require flowing the material through atransparent compartment, thereby extending the use of the semiconductornanocrystal probes to any cytometric method (i.e. any method which isused to detect the presence of detectable substances on the surface orinterior of cells). Instead of flowing the cell-containing materialthrough a transparent compartment, the presence of one or more of aplurality of semiconductor nanocrystal probes bonded to the cells may bedetected by any technique capable of detecting the signals from thedifferent semiconductor nanocrystal probes in a spatially sensitivemanner. Such spatially sensitive detection methods include, for example,confocal microscopy and electron microscopy, as well as theaforementioned flow cytometry.

The following examples will serve to further illustrate the formation ofthe semiconductor nanocrystal probes of the invention, as well as theiruse in detecting the presence of a detectable substance in a materialsuch as a biological material.

EXAMPLE 1

To illustrate the formation of the semiconductor nanocrystal compound(comprising the semiconductor nanocrystals linked to a linking agent) 20ml. of a 5 mM solution of (4-mercapto)benzoic acid was prepared with apH of 10 using (CH₃)₄NOH.5H₂O. 20 mg of tris-octylphosphine oxide coatedCdSe/CdS core/shell nanocrystals were added to the solution and stirreduntil completely dissolved. The resultant nanocrystal/linking agentsolution was heated for 5 hours at 50-60 C and then concentrated to afew ml by evaporation. Then an equal volume of acetone was added and thenanocrystals precipitated out of solution homogeneously. The precipitatewas then washed with acetone, dried, and then can be stored.

The semiconductor nanocrystal compound prepared above can be linked withan appropriate affinity molecule to form the semiconductor nanocrystalprobe of the invention to treat a biological material to determine thepresence or absence of a detectable substance. That is, thesemiconductor nanocrystal compound prepared above can be linked, forexample, with avidin or streptavidin (as the affinity molecule) to forman semiconductor nanocrystal probe to treat a biological material toascertain the presence of biotin; or the semiconductor nanocrystalcompound prepared above can be linked with anti-digoxiginen to form ansemiconductor nanocrystal probe to treat a biological material toascertain the presence of digoxiginen.

EXAMPLE 2

To illustrate the formation of a semiconductor nanocrystal compound(comprising silica coated semiconductor nanocrystals linked to a linkingagent) 200 μl of 3-(mercaptopropyl)-trimethoxysilane and 40 μl of3-(aminopropyl)-trimethoxysilane were added to 120 ml of anhydrous 25%(v/v) dimethylsulfoxide in methanol. The pH of this solution wasadjusted to 10 using 350 μl of a 25% (w/w) solution of (CH₃)₄)NOH inmethanol. 10 mg of CdS or ZnS or ZnS/CdS coated CdSe nanocrystals weredissolved into this solution (prepared, in the case of CdS, by atechnique such as the technique described in the aforementioned Peng,Schlamp, Kadavanich, and Alivisatos article; or in the case of ZdS, bythe technique described by Dabbousi et al. in “(CdSe)ZnS Core-ShellQuantum Dots: Synthesis and Characterization of a Size Series of HighlyLuminescent Nanocrystals,” Journal of Physical Chemistry B 101 pp9463-9475, 1997), stirred to equilibrate for several hours, diluted with200 ml of methanol with 150 μl of a 25% (w/w) solution of (CH₃)₄NOH inmethanol, then heated to boiling for 30 minutes. This solution was thencooled and mixed with a 200 ml solution of 90% (v/v) methanol, 10% (v/v)water, containing 1.0 ml of 3-(trihydroxysilyl)propyl methylphosphonate,monosodium salt (42% w/w solution in water) and 40 μl of3-(aminopropyl)trimethoxysilane. This solution was stirred for twohours, then heated to boiling for fewer than five minutes, then cooled.Once cool, a solution of 4 ml of chlorotrimethylsilane in 36 mlmethanol, the pH of which had been adjusted to 10 using solid(CH₃)₄NOH.5H₂O, was mixed with the solution and stirred for one hour.This solution was then heated to boiling for 30 minutes, cooled to roomtemperature and stirred for several hours more. The solvent wasevacuated partially in vacuo at 60 C. This solution can be precipitatedto an oily solid with acetone. The semiconductor nanocrystal compoundmay then be redissolved in water, and in a variety of buffer solutionsto prepare it for linking it to an affinity molecule to form thesemiconductor nanocrystal probe of the invention to treat a biologicalmaterial to determine the presence or absence of a detectable substance.

Thus, the invention provides an semiconductor nanocrystal probecontaining a semiconductor nanocrystal capable, upon excitation byeither electromagnetic radiation (of either narrow or broad bandwidth)or particle beam, of emitting electromagnetic radiation in a narrowwavelength band and/or absorbing energy and/or scattering or diffractingsaid excitation, thus permitting the simultaneous usage of a number ofsuch probes emitting different wavelengths of electromagnetic radiationto thereby permit simultaneous detection of the presence of a number ofdetectable substances in a given material. The probe material is stablein the presence of light or oxygen, capable of being excited by energyover a wide spectrum, and has a narrow band of emission, resulting in animproved material and process for the simultaneous and/or sequentialdetection of a number of detectable substances in a material such as abiological material.

1.-155. (canceled)
 156. A method of determining a presence of adetectable substance in a material, comprising: contacting the materialwith a semiconductor nanocrystal composition, comprising: a) a corecomprising a first semiconductor material; b) a core-overcoating shellcomprising a second semiconductor material, wherein the core and thecore-overcoating shell form a core/shell nanocrystal; c) a linkingagent; and d) an affinity molecule linked to said core/shell nanocrystalvia the linking agent; wherein said semiconductor nanocrystalcomposition is water-soluble and has a narrow emission wavelength band;and allowing the affinity molecule to selectively bind the detectablesubstance to determine the presence of the substance in the material.157. The method of claim 156, wherein said linking agent forms anencapsulating net around the core/shell nanocrystal.
 158. The method ofclaim 156, wherein said linking agent is polymerizable.
 159. The methodof claim 158, wherein said polymerizable linking agent forms anencapsulating net around the core/shell nanocrystal.
 160. The method ofclaim 156, wherein said narrow wavelength band does not exceed 40 nm inwidth measured at full width half maximum.
 161. The method of claim 156,wherein said narrow wavelength band does not exceed about 20 nm in widthmeasured at full width half maximum.
 162. The method of claim 156,wherein said core has a diameter from about 20 Å to about 100 Å. 163.The method of claim 156, wherein said core-overcoating shell is 1-10monolayers thick.
 164. The method of claim 156, wherein saidcore-overcoating shell epitaxially surrounds said core.
 165. The methodof claim 156, wherein the core/shell nanocrystal has an averagecross-section that is no larger than about 20 nm.
 166. The method ofclaim 156, wherein the core/shell nanocrystal has an averagecross-section of about 1 nm to about 10 nm.
 167. (canceled) 168.(canceled)
 169. The method of claim 156, wherein said firstsemiconductor material comprises a Group II-VI semiconductor.
 170. Themethod of claim 156, wherein said first semiconductor material comprisesa Group III-V semiconductor.
 171. The method of claim 156, wherein saidfirst semiconductor material comprises MgS, MgSe, MgTe, CaS, CaSe, CaTe,SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, or HgTe.
 172. The method of claim 156, wherein said firstsemiconductor material comprises GaAs, InGaAs, InP, or InAs. 173.(canceled)
 174. The method of claim 156, wherein said secondsemiconductor material comprises a Group II-VI semiconductor.
 175. Themethod of claim 156, wherein said second semiconductor materialcomprises a Group III-V semiconductor.
 176. The method of claim 156,wherein said second semiconductor material comprises MgS, MgSe, MgTe,CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS,CdSe, CdTe, HgS, HgSe, or HgTe.
 177. The method of claim 156, whereinsaid second semiconductor material comprises GaAs, InGaAs, InP, or InAs.178. The method of claim 156, wherein said first semiconductor materialcomprises CdSe and the second semiconductor material comprises ZnS. 179.The method of claim 156, wherein said linking agent comprises a thiolmoiety.
 180. The method of claim 156, wherein said linking agentcomprises an alkyl group.
 181. A method of determining a presence of adetectable substance in a material, comprising: contacting the materialwith a semiconductor nanocrystal composition, comprising: a) a corecomprising a first semiconductor material; b) a core-overcoating shellcomprising a second semiconductor material, wherein the core and thecore-overcoating shell form a core/shell nanocrystal; c) a linkingagent; and d) an affinity molecule linked to said core/shell nanocrystalvia the linking agent; wherein said semiconductor nanocrystalcomposition is water-soluble, and has a wavelength emission band notexceeding 40 nm in width, measured at full width half maximum, andwherein the core has an average cross-section no larger than 20 nm; andallowing the affinity molecule to selectively bind the detectablesubstance, if the detectable substance is present in the material. 182.The method of claim 181, wherein said linking agent forms anencapsulating net around the core/shell nanocrystal.
 183. The method ofclaim 181, wherein said linking agent is polymerizable.
 184. The methodof claim 183, wherein said polymerizable linking agent forms anencapsulating net around the core/shell nanocrystal.
 185. (canceled)186. The method of claim 181, wherein said wavelength emission band doesnot exceed about 20 nm in width, measured at full width half maximum.187. The method of claim 181, wherein said semiconductor nanocrystalcomposition has an absorption wavelength band at a shorter wavelengththan the emission wavelength band.
 188. The method of claim 181, whereinsaid core-overcoating shell is 1-10 monolayers thick.
 189. The method ofclaim 181, wherein said core-overcoating shell epitaxially surroundssaid core.
 190. The method of claim 181, wherein the core/shellnanocrystal has an average cross-section that is no larger than 10 nm.191. The method of claim 181, wherein the core/shell nanocrystal has anaverage cross-section of about 1 nm to about 10 nm.
 192. (canceled) 193.(canceled)
 194. The method of claim 181, wherein said firstsemiconductor material comprises a Group II-VI semiconductor.
 195. Themethod of claim 181, wherein said first semiconductor material comprisesa Group III-V semiconductor.
 196. The method of claim 181, wherein saidfirst semiconductor material comprises MgS, MgSe, MgTe, CaS, CaSe, CaTe,SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, or HgTe.
 197. The method of claim 181, wherein said firstsemiconductor material comprises GaAs, InGaAs, InP, or InAs. 198.(canceled)
 199. The method of claim 181, wherein said secondsemiconductor material comprises a Group II-VI semiconductor.
 200. Themethod of claim 181, wherein said second semiconductor materialcomprises a Group III-V semiconductor.
 201. The method of claim 181,wherein said second semiconductor material comprises MgS, MgSe, MgTe,CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS,CdSe, CdTe, HgS, HgSe, or HgTe.
 202. The method of claim 181, whereinsaid second semiconductor material comprises GaAs, InGaAs, InP, or InAs.203. The method of claim 181, wherein said first semiconductor materialcomprises CdSe and the second semiconductor material comprises ZnS. 204.The method of claim 181, wherein said linking agent comprises a thiolmoiety.
 205. The method of claim 181, wherein said linking agentcomprises an alkyl group.
 206. The method of claim 156, furthercomprising: exposing the semiconductor nanocrystal composition to afirst electromagnetic energy; and detecting emission of a secondelectromagnetic energy from the semiconductor nanocrystal composition.207. The method of claim 156, wherein the detectable substance is abiological substance.
 208. The method of claim 156, further comprising:after allowing the affinity molecule to selectively bind the detectablesubstance, if the detectable substance is present in the material,separating away the material from the semiconductor nanocrystalcomposition.
 209. The method of claim 156, wherein said linking agent isadhered to the core/shell nanocrystal through Van der Waals' forces.210. The method of claim 156, wherein the linking agent is adhered tothe core/shell nanocrystal through a bond selected from the groupconsisting of hydrogen bonding, mechanical bonding, covalent bonding andionic bonding.
 211. The method of claim 156, wherein the linking agentis a polymer.
 212. The method of claim 211, wherein the polymer forms anencapsulating net around the core/shell nanocrystal.
 213. The method ofclaim 156, wherein the linking agent is selected from diacetylenes,acrylates, acrylamides, vinyl, styryl, silicon oxide, boron oxide,phosphorus oxide, silicates, borates and phosphates.
 214. The method ofclaim 181, further comprising: exposing the semiconductor nanocrystalcomposition to a first electromagnetic energy; and detecting emission ofa second electromagnetic energy from the semiconductor nanocrystalcomposition.
 215. The method of claim 181, further comprising: afterallowing the affinity molecule to selectively bind the detectablesubstance, if the detectable substance is present in the material,separating away the material from the semiconductor nanocrystalcomposition.
 216. The method of claim 181, wherein the detectablesubstance is a biological substance.
 217. The method of claim 181,wherein said linking agent is adhered to the core/shell nanocrystalthrough Van der Waals' forces.
 218. The method of claim 181, wherein thelinking agent is a polymer.
 219. The method of claim 181, wherein thelinking agent is selected from diacetylenes, acrylates, acrylamides,vinyl, styryl, silicon oxide, boron oxide, phosphorus oxide, silicates,borates and phosphates.
 220. A method of producing a luminescentsemiconductor nanocrystal composition, comprising: providing asemiconductor core comprising a first semiconductor material selectedfrom the group consisting of a II-VI semiconductor and a III-Vsemiconductor; depositing an overcoating shell on the core, wherein saidcore-overcoating shell comprises a second semiconductor material whichis different from the first semiconductor material, the secondsemiconductor material being selected from the group consisting of MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe, and wherein the coreand the core-overcoating shell form a core/shell nanocrystal; andassociating a polymer with the core/shell nanocrystal to produce aluminescent semiconductor nanocrystal composition.
 221. The method ofclaim 220, wherein the polymer is associated with the core/shellnanocrystal by mixing the core/shell nanocrystal with the polymer. 222.The method of claim 220, wherein the polymer is covalently bound to thesecond semiconductor material.
 223. The method of claim 220, wherein thecore/shell nanocrystal is incorporated into the interior of the polymer.224. The method of claim 220, wherein the polymer is associated with thecore/shell nanocrystal by adsorption.
 225. The method of claim 220,wherein the first semiconductor material is selected from the groupconsisting of MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe. 226.The method of claim 220, wherein said first semiconductor material isselected from the group consisting of GaAs, InGaAs, InP, and InAs. 227.The method of claim 220, wherein said second semiconductor material isselected from the group consisting of CdSe and ZnSe.
 228. The method ofclaim 220, wherein the first semiconductor material is CdSe and thesecond semiconductor material is ZnS.
 229. The method of claim 220,wherein the core has a diameter in a range of from about 20 Å to about100 Å.
 230. The method of claim 220, wherein the core-overcoating shellcomprises 1 to 10 monolayers.
 231. The method of claim 220, wherein thecore-overcoating shell comprises ZnS and wherein the polymerencapsulates the core/shell nanocrystal.
 232. The method of claim 220,wherein the composition emits radiation within a narrow wavelength bandof about 40 nm or less in width, measured at full width half maximum.233. The method of claim 156, wherein the linking agent is3-aminopropyl-trimethoxy silane.
 234. The method of claim 156, whereinthe linking agent is 3-mercaptopropyl-trimethoxy silane.
 235. The methodof claim 156, wherein the linking agent comprises a phosphate moiety.236. The method of claim 181, wherein the linking agent is3-aminopropyl-trimethoxy silane.
 237. The method of claim 181, whereinthe linking agent is 3-mercaptopropyl-trimethoxy silane.
 238. The methodof claim 181, wherein the linking agent comprises a phosphate moiety.